The present invention encompasses novel analogs of vasoactive intestinal peptide (VIP), containing substitutions at appropriately selected amino acids. The invention particularly relates to the design and synthesis of novel biologically active VIP analogs containing xcex1,xcex1-dialkylated amino acids in a site-specific manner. Specifically, the invention relates to the synthesis of VIP peptide derivatives, which bind selectively to VIP receptors on target cells. The invention encompasses methods for the generation of these peptides, compositions containing the peptides and the pharmacological applications of these peptides especially in the treatment and prevention of cancer.
Vasoactive intestinal peptide is known to play critical roles in modulating the intracellular and extracellular events involved in homeostasis, and are intimately involved in all major cognitive and non-cognitive homeostatic systems (Schofl et al. 1994). The multiple biological activities of peptides has led to extensive research focused on the exploitation of these peptide hormones as therapeutic drugs. Multiple replacements have been used to avoid the susceptibility of the amide bond to proteolytic cleavage. These include the use of nonstandard amino acids like D-amino acids, N-alkyl and N-hydroxy-amino acids, xcex1-aza amino acids, thioamide linkage, design of peptide mimetics and prodrugs as well as amide bond modifications under the pseudopeptide linkage rubric (Dutta, 1993; Pasternak et al., 1999). Another approach has been the blockage of N-terminus or C-terminus of the peptide by acylation or amidation.
Vasoactive intestinal peptide is a 28-amino acid neuropeptide, which was first isolated from the porcine intestine (Said and Mutt, 1970). It bears extensive homology to secretin, peptide histidine isoleucine (PHI) and glucagon.
The amino acid sequence for VIP is
His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (SEQ ID NO: 1).
VIP is known to exhibit a wide variety of biological activities in the autocrine, endocrine and paracrine functions in living organisms (Said, 1984). In the gastrointestinal tract, it has been known to stimulate pancreatic and biliary secretions, hepatic glycogenesis as well as the secretion of insulin and glucagon (Kerrins and Said, 1972; Domschke et al., 1977). In the nervous system it acts as a neurotransmitter and neuromodulator, regulating the release and secretion of several key hormones (Said, 1984). In recent years, attention has been focussed on the function of VIP in certain areas of the CNS as well its role in the progression and control of neoplastic disease (Reubi, 1995).
The importance of peptide growth factors and regulatory hormones in the etiology and pathogenesis in several carcinomas has long been recognized. Data from epidemiological and endocrinological studies suggest that neuropeptides like VIP which are responsible for the normal growth of tissues like the pancreas can also cause conditions for their neoplastic transformation (Sporn et al., 1980). Several lines of evidence indicate that VIP acts as a growth factor and plays a dominant autocrine and paracrine role in the sustained proliferation of cancer cells (Said, 1984). The stimulatory effect of VIP on tumor growth can be mediated directly by its receptors on cell membranes or indirectly by potentiation of the activities of other growth factors in tumor cells (Scholar et al., 1991). The synergistic effect of VIP and related pituitary adenylate cyclase activating polypeptide (PACAP) in glioblastomas is an illustration to the above fact (Moody et al., 1996).
The multiple physiological and pharmacological activities of VIP are mediated by high affinity G-protein coupled transmembrane receptors on target cells (Reubi et al., 1996). VIP receptors are coupled to cellular effector systems via adenyl cyclase activity (Xia et al., 1996). The VIP receptor, found to be highly over expressed in neoplastic cells, is thought to be one of the biomarkers in human cancers (Reubi et al., 1996). High affinity VIP receptors have been localized and characterized in neoplastic cells of most breast carcinomas, breast and prostate cancer metastases, ovarian, colonic and pancreatic adenocarcinomas, endometrial and squamous cell carcinomas, non small cell lung cancer, lymphomas, glioblastomas, astrocytomas, meningiomas and tumors of mesenchymal origin. Amongst, neuroendocrine tumors all differentiated and non-differentiated gastroenteropancreatic tumors, pheochromocytomas, small-cell lung cancers, neuroblastomas, pituitary adenomas as well tumors associated with hypersecretory states like Verner-Morrison syndrome were found to overexpress receptors for vasoactive intestinal peptide (Tang et al., 1997a and b; Moody et al., 1998a andb; Oka et al., 1998)). These findings suggest that new approaches for the diagnosis and treatment of these cancers may be based on functional manipulation of VIP activity, by designing suitable peptide derivatives of the same.
The present invention relates to the anti-neoplastic activity of novel VIP peptide analogs using selected constrained amino acids. These novel VIP analogs were found to bind to VIP receptor on cell membranes. The anti-neoplastic activity of the aforesaid peptides was also determined.
The design of confornationally constrained bioactive peptide derivatives has been one of the widely used approaches for the development of peptide-based therapeutic agents. Non-standard amino acids with strong conformational preferences may be used to direct the course of polypeptide chain folding, by imposing local stereochemical constraints, in de novo approaches to peptide design. The conformational characteristics of xcex1,xcex1-dialkylated amino acids are have been well studied. The incorporation of these amino acids restricts the rotation of xcfx86, xcexa8, angles, within the molecule, thereby stabilizing a desired peptide conformation. The prototypic member of xcex1,xcex1-dialkylated aminoacids, xcex1-amino-isobutyric acid (Aib) or xcex1,xcex1-dimethylglycine has been shown to induce (xcex2-turn or helical conformation when incorporated in a peptide sequence (Prasad and Balaram, 1984, Karle and Balaram, 1990). The conformational properties of the higher homologs of xcex1,xcex1-dialkylated amino acids such as di-ethylglycine (Deg), di-n-propylglycine (Dpg), di-n-butylglycine (Dbg) as well as the cyclic side chain analogs of xcex1,xcex1-dialkylated amino acids such as 1-aminocyclopentane carboxylic acid (Ac5c), 1-aminocyclohexane carboxylic acid (Ac6c), 1-aminocycloheptane carboxylic acid (Ac7c) and 1-aminocyclooctane carboxylic acid (Ac8c) have also been shown to induce folded conformation (Prasad et al., 1995; Karle et al., 1995). xcex1,xcex1-dialkylated amino acids have been used in the design of highly potent chemo-tactic peptide analogs (Prasad et al., 1996). The applicants are not aware of any prior art for the synthesis of novel peptide analogs, encompassed in the present invention. The present invention exploits the conformational properties of xcex1,xcex1-dialkylated amino acids for the design of biologically active peptide derivatives, taking VIP as the model system under consideration.
Domschke, S. et al. (1977) Gastroenterology, 73, 478-480.
Dutta, A. S. (1993) Small Peptides: Chemistry, Biology and Clinical Studies, Elsevier,
Pharmacochemistry Library, 19, pp 293-350.
Karle, I. L. et al. (1995) J. Amer. Chem. Soc. 117, 9632-9637.
Karle, I. L. and Balaram, P. (1990) Biochemistry 29, 6747-6756.
Kerrins, C. and Said, S. I. (1972) Proc. Soc. Exp. Biol. Med. 142, 014-1017.
Oka, H. et al. (1998) Am. J. Pathol. 153, 1787-1796.
Pasternak, A. et al. (1999) Biorg. Med. Chem. 9, 491-496.
Prasad, B V V and Balaram, P. (1984) CRC Crit. Rev. Biochemn. 16, 307-347.
Prasad, S et al. (1995) Biopolymers 35, I 1-20
Prasad, S et al. (1996) Int. J. Peptide Protein Res. 48, 312-318.
Reubi, J. C. et al. Cancer Res., 56 (8), 1922-1931, 1996.
Said, S. I. and Mutt, V. (1970) Science, 169, 1217-1218.
Said, S. I. (1984) Peptides, 5, 143-150.
Scholar E. M. Cancer 67(6): 1561-1569, 1991.
Scofi, C. et al. (1994) Trends. Endocrinol. Metab. 5, 53-59.
Sporn, M. B., and Todaro, G. J. (1980) N. Engl. J. Med., 303, 378-379.
Stewart, J. and Young , Y. D. (1969) Solid Phase Peptide Synthesis, W. H. Freeman and Co.
Tang, C. et al., (1997a) Gut, 40, 267-271.
Tang, C. et al., (1997b) Br. J. Cancer, 75, 1467-1473.
Xia, M. et al., J. Clin. Immunol., 16 (1), 21-30, 1996
Throughout the specification and claims the following abbreviations are used with the following meanings:
BOP: Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexfluorophosphate
PyBOP: Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexofluorophospate
HBTU: O-Benzotriazole-N,N,Nxe2x80x2,Nxe2x80x2-tetramethyl-uronium-hexofluoro-phosphate
TBTU: 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetra-methyluronium tetrafluoroborate
HOBt: 1-Hydroxy Benzotriazole
DCC: Dicyclohexyl carbodiimide
DIPCDI: Diisopropyl carbodiimide
DIEA: Diisopropyl ethylamine
DMF: Dimethyl formamide
DCM: Dichloromethane
NMP: N-Methyl-2-pyrrolidinone
TFA: trifluoroacetic acid
Throughout the specification and claims the amino acid residues are designated by their standard abbreviations. Amino acids denote L-configuration unless otherwise indicated by D or DL appearing before the symbol and separated from it by a hyphen.
The present invention comprises VIP antagonists of the following general formula, wherein appropriate amino acids in VIP have been replaced by xcex1,xcex1-dialkylated amino acids in a specific manner. The invention also comprises the pharmaceutically acceptable salts of the antagonists of the following general formula:
His-Ser-Asp-R1-Val-R2-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-R3-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2
wherein
R1 is Aib, Deg or Ac5c,
R2 is Phe or 4-Cl-D-Phe,
R3 is Met, Leu or Dpg or a hydrolyzable carboxy protecting group;
or pharmaceutically acceptable salt of the antagonists.
The present invention comprises VIP antagonists of the following general formula, wherein appropriate amino acids in VIP have been replaced by xcex1,xcex1-dialkylated amino acids in a specific manner. The invention also comprises the pharmaceutically acceptable salts of the antagonists of the following general formula:
His-Ser-Asp-R1-Val-R2-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-R3-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2
wherein
R1 is Aib, Deg or Ac5c,
R2 is Phe or 4-Cl-D-Phe,
R3 is Met, Leu or Dpg or a hydrolyzable carboxy protecting group;
or pharmaceutically acceptable salt of the antagonists.
A hydrolyzable carboxy protecting group are those groups which on hydrolysis converts to carboxy group such as xe2x80x94CONH2, xe2x80x94COOMe, etc.
Salts encompassed within the term xe2x80x9cpharmaceutically acceptable saltsxe2x80x9d refer to non-toxic salts of the compounds of this invention. Representative salts and esters include the following:
acetate, ascorbate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, caamsylate, carbonate, citrate, dihydrochloride, methanesulfonate, ethanesulfonate, xcfx81-toluenesulfonate, cyclohexylsulfamate, quinate, edetate, edisylate, estolate, esylate, fumarate, gluconate, glutamate, glycerophophates, hydrobromide, hydrochloride, hydroxynaphthoate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, mucate, napsylate, nitrate, n-methylglucamine, oleate, oxalate, palmoates, pamoate (embonate), palmitate, pantothenate, perchlorates, phosphate/diphosphate, polygalacturonate, salicylates, stearate, succinates, sulfate, sulfamate, subacetate, succinate, tannate, tartrate, tosylate, trifluoroacetate, and valerate.
Other salts include Ca, Li, Mg, Na, and K salts; salts of amino acids such as lysine or arginine; guanidine, diethanolamine or choline; ammonium, substituted ammonium salts or aluminum salts.
The salts are prepared by conventional methods.
The preferred VIP antagonists of the present invention are as follows:
(R1=Aib, R2=4-D-Cl-Phe, and R3=Leu):
His-Ser-Asp-Aib-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (SEQ ID NO: 2);
(R1=Deg, R2=4-Cl-D-Phe, and R3=Leu):
His-Ser-Asp-Deg-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (SEQ ID NO: 3);
(R1=Ac5c, R2=4-Cl-D-Phe, and R3=Leu):
His-Ser-Asp-Ac5c-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (SEQ ID NO: 4);
(R1=Aib, R2=Phe, and R3=Met):
His-Ser-Asp-Aib-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (SEQ ID NO: 5);
(R1=Aib, R2=Phe, and R3=Leu):
His-Ser-Asp-Aib-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2, (SEQ ID NO: 6);
(R1=Ac5c, R2=Phe, and R3=Leu):
His-Ser-Asp-Ac5c-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (SEQ ID NO: 7);
(R1=Deg, R2=Phe, and R3=Leu):
His-Ser-Asp-Deg-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (SEQ ID NO: 8);
(R1=Aib, R2=Phe, and R3=Dpg):
His-Ser-Asp-Aib-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Dpg-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (SEQ ID NO: 9);
(R1=Aib, R2=4-Cl-D-Phe, and R3=Dpg):
His-Ser-Asp-Aib-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Dpg-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (SEQ ID NO: 10);
(R1=Deg, R2 Phe, and R3 Dpg):
His-Ser-Asp-Deg-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Dpg-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (SEQ ID NO: 11);
(R1=Ac5c, R2=Phe, and R3=Dpg):
His-Ser-Asp-Ac5c-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Dpg-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (SEQ ID NO: 12).
The novel compounds of the present invention have important pharmacological applications. They are potent anti-neoplastic agents and thereby possess therapeutic potential in a number of human cancers.
Suitable routes of administration are those known in the art and include oral, rectal, transdermal, vaginal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
Pharmaceutical compositions suitable for use in present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. The term xe2x80x9can effective amountxe2x80x9d means that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal or human that is being sought.
In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers excipients, diluents, solvents, flavorings, colorants etc. The preparations may be formulated in any form including but not limited to tablets, dragees, capsules, powders, syrups, suspensions, slurries, time released formulations, sustained release formulations, pills, granules, emulsions, patches, injections, solutions, liposomes and nanoparticles.
The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient""s condition.
Toxicity and therapeutic efficacy of the peptides of this invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals.
The novel peptide analogs embodied in the present invention contain amino acids, namely xcex1,xcex1-dialkylated amino acids, which have been known to induce highly specific constraints in the peptide backbone.
The xcex1,xcex1-dialkylated amino acids, used in the present invention are synthesized from the corresponding ketones. In a preferred embodiment of the invention, the ketones are first converted into the corresponding hydantoins, which are hydrolyzed using a strong acid or base, preferably H2SO4, HCl, NaOH, or Na2CO3 to yield the aforesaid amino acids. In a preferred embodiment of the present invention, 60% sulphuric acid has been employed as the hydrolyzing agent. The conversion of the ketones to their appropriate xcex1,xcex1-dialkylated amino acids is shown in Example 1.
The novel peptides in the present invention have been generated by using solid phase techniques or by a combination of solution phase procedures and solid phase techniques or by fragment condensation (Stewart and Young, 1969).
In a preferred embodiment of the present invention the peptides were synthesized using the Fmoc strategy, on a semi automatic peptide synthesizer (CS Bio, Model 536), using optimum side chain protection. The peptides were assembled from C-terminus to N-terminus. Peptides amidated at the carboxy-terminus were synthesized using the Rink Amide resin. The loading of the first Fmoc protected amino acid was achieved via an amide bond formation with the solid support, mediated by Diisopropylcarbodiimide (DIPCDI) and HOBt. Substitution levels for automated synthesis were preferably between 0.2 and 0.6 mmole amino acid per gram resin. The steps involved in the synthesis of the VIP analogs employed the following protocol:
The resin employed for the synthesis of carboxy-terminal amidated peptide analogs was 4-(2xe2x80x2,4xe2x80x2-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxymethyl derivatized polystyrene 1% divinylbenzene (Rink Amide) resin (100-200 mesh), procured from Calbioichem-Novabiochem Corp., La Jolla, U.S.A., (0.47 milliequivalent NH2/g resin).
The present invention also provides a solid phase synthesis process for the preparation of a peptide analog of formula (I):
His-Ser-Asp-R1-Val-R2-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-R3-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2
wherein
R1 is Aib, Deg or Ac5c,
R2 is Phe or 4-Cl-D-Phe,
R3 is Met, Leu or Dpg,
which comprises sequentially loading the corresponding protected xcex1xcex1-dialkylated amino acids in sequential cycles to the amino terminus of a solid phase resin, coupling the amino acids in the presence of conventional solvents and reagents to assemble a peptide-resin assembly, removing the protecting groups and cleaving the peptide from the resin to obtain a crude peptide analog.
In a particularly preferred embodiment of the present invention the following chemical moieties were used to protect reactive side chains of the peptides during the synthesis procedure.
The N-terminal amino group was protected by 9-fluorenylnethoxy-carbonyl group. Trityl (trt) or t-butyloxycarbonyl (Boc) were the preferred protecting groups for imadazole group of Histidine residue. The hydroxyl groups of Serine, Threonine and Tyrosine were preferably protected by t-butyl group (tBu) 2,2,5,7,8-pentamethyl-chroman-6-sulfonyl (Pmc) or 2,2,4,7,-pentamethyl-dihydrobenzenofuran-5-sulfonyl (Pbf) were the preferred protecting groups for the guandino group of Arginine. Trityl was the preferred protecting group for Asparagine and Glutamine and tertiary butyl group (tBu) was the preferred protecting group for Aspartic acid and Glutamic acid. The tryptophan residue was either left unprotected or used with Boc protection. The side chain amino group of Lysine was protected using preferably Boc group.
In a preferred embodiment of the invention, 2-8 equivalents of Fmoc protected amino acid per resin nitrogen equivalent were used. The activating reagents used for coupling amino acids to the resin, in solid phase peptide synthesis, are well known in the art. These include DCC, DIPCDI, DIEA, BOP, PyBOP, HBTU, TBTU, and HOBt. Preferably, DCC or DIPCDI/HOBt or HBTU/HOBT and DIEA were used as activating reagents in the coupling reactions.
The protected amino acids were either activated in situ or added in the form of preactivated esters known in the art such as NHS esters, Opfp esters etc therton, E. et al., 1988, J. Chem.Soc., Perkin Trans. I, 2887; Bodansky, M in xe2x80x9cThe Peptides, Analysis, Synthesis and Biology (E. Gross, J, Meienhofer,eds) Vol. 1, Academic Press, New York, 1979, 106.
The coupling reaction was carried out in DMF, DCM or NMP or a mixture of these solvents and was monitored by Kaiser test (Kaiser et al., Anal. Biochem., 34, 595-598 (1970)). In case of a positive Kaiser test, the appropriate amino acid was re-coupled using freshly prepared activated reagents.
After the assembly of the peptide analog was completed, the amino-terminal Fmoc group was removed using steps 1-6 of the above protocol and then the peptide resin was washed with methanol and dried. The analogs were then deprotected and cleaved from the resin support by treatment with a cleavage mixture of trifluoroacetic acid, crystalline phenol, ethanedithiol, thioanisole and de-ionized water for 1.5 to 5 hours at room temperature. The crude peptide was obtained by precipitation with cold dry ether, filtered, dissolved, and lyophilized.
The resulting crude peptide was purified by preperative high performance liquid chromatography (HPLC) using a LiChrOCART(copyright) C18 (250. Times. 10) reverse phase column (Merck, Darmstadt, Germany) on a Preparative HPLC system (Shimadzu Corporation, Japan) using a gradient of 0.1% TFA in acetonitrile and water. The eluted fractions were reanalyzed on Analytical HPLC system (Shimadzu Corporation, Japan) using a C18 LiChrospher(copyright), WP-300 (300xc3x974) reverse-phase column. Acetonitrile was evaporated and the fractions were lyophilized to obtain the pure peptide. The identity of each peptide was confirmed by electron-spray mass spectroscopy.
An analog of the present invention can be made by exclusively solid phase techniques, by partial solid phase/solution phase techniques and/or fragment condensation. Preferred, semi-automated, stepwise solid phase methods for synthesis of peptides of the invention are provided in the examples discussed in the subsequent section of this document.